Calcium dependence of NKT-cell responses. (A) NKT cells (clone J24L.17) were stimulated by CD1d transfectants that were either untreated (filled symbols) or pulsed with the indicated concentrations of α-GalCer (open symbols). Cytokine secretion and calcium flux responses were normalized by the response to 100 ng/mL α-GalCer. Similar results were observed for 2 additional NKT cell clones. (B) NKT cells were stimulated by α-GalCer–pulsed CD1d transfectants in the presence of titrated doses of cyclosporin A (CsA, left plot), or the MEK1/2 inhibitor U0126 (right plot) and GM-CSF and IL-2 were quantitated by ELISA. The half-maximal effective concentrations (EC₅₀) of CsA for GM-CSF and IL-2 were calculated by nonlinear regression using a sigmoidal dose-response equation. The 95% confidence intervals for inhibition by CsA were as follows: IL-2, 14.07 to 18.71 nM; GM-CSF, 108.8 to 134.6 nM. For inhibition by U0126, they were: IL-2, 0.797 to 1.135 μM; GM-CSF, 2.000 to 3.636 μM. (C) NKT cells alone or in the presence of the indicated APCs were treated with 100 nM of ionomycin (■) or vehicle (□), and cytokine secretion was quantitated by ELISA. Left plot shows GM-CSF; right plot shows IL-2. (D) NKT-cell calcium flux after stimulation by APCs alone or in the presence of 100 nM of ionomycin. The results shown in panels B to D are each from 1 representative experiment of 3 using clone J24N.22. Similar results were observed using 2 different NKT cell clones.